Metabolic screening using on-line ultrafiltration mass spectrometry.
An on-line mass spectrometric method has been developed to generate and identify drug metabolites formed by hepatic cytochromes P450. This method, pulsed ultrafiltration-mass spectrometry, may be used for rapid screening of drugs to determine their extent of metabolism by microsomal cytochromes P450 and to characterize the primary metabolites. Rat liver microsomes were trapped in a stirred ultrafiltration chamber fitted with a 100,000 molecular weight cut-off ultrafiltration membrane. A continuous-flow of ammonium acetate buffer was pumped through the chamber and into an electrospray mass spectrometer. Substrates for cytochromes P450 including imipramine, chlorpromazine, and pentoxyresorufin were flow injected through the chamber along with the cofactor, NADPH, and metabolites were detected on-line by using electrospray mass spectrometry. Identical control experiments carried out using boiled microsomes or without NADPH showed no metabolite formation. Naringenin and quinidine, which are inhibitors of some isozymes of cytochrome P450 and are not known to be extensively metabolized, showed no major metabolites. For comparison, imipramine metabolites were also generated by standard batch incubation with microsomes and NADPH, followed by extraction and LC-MS analysis. Similar metabolites were obtained using the flow-through ultrafiltration method and the standard batch microsomal incubation. Tandem mass spectrometry was used to confirm structures of imipramine metabolites including 10-hydroxyimipramine, 2-hydroxyimipramine, imipramine N-oxide, and N-desmethylimipramine. Finally, the feasibility of using ultrafiltration mass spectrometry for high throughput metabolic screening was demonstrated by using on-line mass spectrometry for only 3 min per incubation instead of monitoring the entire elution profile. By carrying out multiple ultrafiltration experiments in parallel, efficient use of the mass spectrometric detector may be obtained with a throughput of at least 20 incubations per hour. Throughputs of up to 60 profiles per hour should be possible. On-line ultrafiltration electrospray mass spectrometry offers a streamlined, higher-throughput method for in vitro formation and mass spectrometric characterization of microsomal drug metabolites.
van Breemen
R B
RB
Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612-7231, USA. richard.vanbreemen@uic.edu
Nikolic
D
D
Bolton
J L
JL
D000818:Animals; D001331:Automation; D002746:Chlorpromazine / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005260:Female; D044950:Flavanones; D005419:Flavonoids / Q000378:metabolism; D007099:Imipramine / Q000378:metabolism; D008658:Inactivation, Metabolic / Q000502:physiology*; D008297:Male; D013058:Mass Spectrometry / Q000295:instrumentation; D008862:Microsomes, Liver / Q000201:enzymology*; D010078:Oxazines / Q000378:metabolism; D011802:Quinidine / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014462:Ultrafiltration / Q000295:instrumentation
D044950:Flavanones; D005419:Flavonoids; D010078:Oxazines; C044943:pentoxyresorufin; D003577:Cytochrome P-450 Enzyme System; C005273:naringenin; D011802:Quinidine; D007099:Imipramine; D002746:Chlorpromazine